The Na-K-CI cotransporter (NKCC) is a plasma membrane transport protein that plays a central role in cellular homeostasis. In non-polarized cells the NKCC1 isoform is involved in regulation of cell volume. In secretory epithelia, NKCC1 functions together with CI channels, the Na pump, and K channels to bring about regulated salt movement. In the mammalian kidney another isoform, NKCC2, mediates salt absorption and is the site of action of the loop diuretic drugs furosemide and bumetanide. NKCCs are members of the cation-chloride cotransporter family, which also includes Na-CI and K-CI cotransporters. Previous work from this laboratory has resulted in the cloning and characterization of many of the members of this family including the NKCCs. The long term goal of this project is to understand the molecular mechanism of the cotransporter, including the structural and functional features underlying ion translocation and its regulation, and the significance of the transporter in cell and organ function. The Specific Aims of the project are: 1) To examine structure-function relationships in ion and bumetanide binding using site-directed mutants of shark and human NKCC1 expressed in HEK-293 cells: test the hypothesis that serines and threonines are involved in the ion transport pocket, and examine the topology model. 2) To determine the mechanism of regulation of NKCC1: find and charactere phosphorylation sites, isolate the regulatory kinase, uncover the mechanism of CI dependency of regulation, and examine the role of a phosphatase binding site. These experiments will utilize isolated shark rectal gland cells as well as NKCC1 constructs in HEK cells. 3) To clone and characterize a third isoform of the Na-K-CI cotransporter, NKCC3, and determine the cells and tissues in which it is expressed. 4) To directly test hypotheses concerning the physiological role of NKCC1 in tissues such as lung, mammary gland, kidney, and red cells by utilizing Cre recombinase- mediated tissue-specific knockouts in a NKCC/loxP mouse model.